Neurotoxin formulation

ABSTRACT

The invention relates to stable liquid neurotoxin formulations which are free of animal proteins, comprising a surfactant, an amino acid selected from tryptophan and tyrosine, a buffer comprising sodium, chloride and phosphate ions, which have a pH between 5.5 and 8, and which are stable for 2 months. These compositions are suitable for use in therapy and in particular for administration to a patient to achieve a desired therapeutic or aesthetic effect. The invention also relates to the use of an amino acid selected from tryptophan and tyrosine to protect a proteinaceous neurotoxin from degradation in a liquid composition which is free of animal derived proteins.

The present invention relates to animal protein free liquid neurotoxinformulations. In particular, the present invention relates to animalprotein free liquid botulinum neurotoxin formulations stabilized withnon proteinaceous excipients.

The neurotoxin formulations described herein are suitable for use intherapy and in particular for administration to a patient to achieve adesired therapeutic or aesthetic effect.

BACKGROUND OF THE INVENTION

Clostridial neurotoxins naturally produced by clostridial strains arethe most toxic biological agents known to date and at the same time arepowerful tools for the treatment of a number of neuromuscular andendocrine disorders, including cervical dystonia, spasticity,blepharospasm, hyperhidrosis or sialorrhea. They also find uses in theaesthetic field for the smoothing of wrinkles.

In order to be suitable for use as a pharmaceutical product, aneurotoxin composition must be such that it can be stored withoutsignificant loss of neurotoxin activity.

In all currently approved formulations of botulinum neurotoxins, ananimal (including human) protein, usually human serum albumin (HSA), isused as a stabiliser.

The presence of animal proteins such as HSA in pharmaceuticalcompositions is however undesirable because of the risk, even if low, ofunwillingly transmitting animal borne infectious agents such as prionsto a patient.

Animal protein free botulinum toxin formulations have been disclosed inthe art. For example, WO0158472 describes lyophilized compositions inwhich a polysaccharide, such as 2-hydroxyethyl starch is used tostabilize a botulinum toxin. WO2005007185 describes compositions inwhich a surface active substance, and a mixture of at least two aminoacids selected from Glu and Gln or Asp and Asn are used to stabilize abotulinum toxin.

Most prior art formulations are however not stable in liquid form andare therefore stored in lyophilized or freeze-dried form. Suchformulations need to be reconstituted by the physician in a sterilesaline solution before administration to a patient. This reconstitutionstep is associated with a loss of physician time, a risk of a dilutionerror and also a risk of contamination during the reconstitutionprocess. The botulinum toxin provider must also train the physicians inorder to ensure that the reconstitution step is performed adequately.

Liquid formulations are therefore advantageous as they obviate the lossof time for the physician, the risk of a dilution error, thecontamination risk and the need for providing training for the provider.

Liquid HSA-free formulations are described for example in WO2006005910which discloses liquid botulinum toxin formulations comprising asurfactant, sodium chloride and a disaccharide. WO2009008595 disclosesliquid botulinum toxin formulations comprising polysorbate 20 andmethionine.

It is an objective of the present invention to provide advantageousliquid animal protein free botulinum neurotoxin formulations, which aresuitable for storage and for use in therapy. In particular, thestabilizing formulation should maintain product stability, be free ofanimal proteins and also be suitable for stabilising a neurotoxin whichis free of complexing proteins.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a liquid compositioncomprising or consisting essentially of a proteinaceous neurotoxin, asurfactant, an amino acid selected from tryptophan and tyrosine, abuffer comprising sodium, chloride and phosphate ions, which has a pHbetween 5.5 and 8, which is stable over time and which is free of animalderived proteins.

Another aspect is the use of the liquid compositions according to theinvention in therapy and/or in cosmetics.

A further aspect of the present invention is the use of an amino acidselected from tryptophan and tyrosine to protect a proteinaceousneurotoxin from degradation in a liquid composition which is free ofanimal derived proteins.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is a liquid compositioncomprising or consisting essentially of a proteinaceous neurotoxin, asurfactant, an amino acid selected from tryptophan and tyrosine, abuffer comprising sodium, chloride and phosphate ions, which has a pHbetween 5.5 and 8, which is stable over time and which is free of animalderived proteins.

“Animal protein free” is to be understood as comprising no protein ofanimal, including human, origin.

A neurotoxin is a substance that targets a nerve cell and affects aneurological function. Proteinaceous neurotoxins include botulinumtoxins (BoNT) and tetanus toxin (TeNT). Preferably, the proteinaceousneurotoxin is a botulinum neurotoxin.

Botulinum neurotoxins are 150 kDa metalloproteases that consist in theiractive form of a 50 kDa light chain (L) and a 100 kDa heavy chain (H)linked by a disulfide bridge. The L chain is a zinc-protease whichintracellularly cleaves one of the SNARE (Soluble NSF Attachment ProteinREceptor) proteins involved in vesicle mediated neurotransmitterrelease, thereby disrupting neurotransmitter mediated mechanisms. Theheavy chain encompasses two domains: an N-terminal 50 kDa translocationdomain (H_(N)), and a C-terminal 50 kDa receptor-binding domain (H_(C)).The H_(C) domain of a botulinum neurotoxin comprises two distinctstructural features that are referred to as the H_(CC) and H_(CN)domains. Amino acid residues involved in receptor binding are believedto be primarily located in the H_(CC) domain.

Botulinum neurotoxins have been classified in 7 antigenically distinctserotypes (A to G). Exemplary amino acid sequences for each serotype areprovided herein as SEQ ID NO 1 to 7.

For each of the sequences, the different domains can for example be asfollow.

H_(CN) H_(CC) Serotype L chain H_(N) domain domain domain BoNT/A (SEQ IDNO 1) 1-448 449-871 872-1110 1111-1296 BoNT/B (SEQ ID NO 2) 1-440441-858 859-1097 1098-1291 BoNT/C (SEQ ID NO 3) 1-441 442-866 867-11111112-1291 BoNT/D (SEQ ID NO 4) 1-445 446-862 863-1098 1099-1276 BoNT/E(SEQ ID NO 5) 1-422 423-845 846-1085 1086-1252 BoNT/F (SEQ ID NO 6)1-439 440-864 865-1105 1106-1274 BoNT/G (SEQ ID NO 7) 1-441 442-863864-1105 1106-1297

The skilled person will appreciate that there can be some variation ineach of the botulinum neurotoxin domains.

BoNTs act for example on neuromuscular nerve junctions by preventingrelease of acetylcholine and thereby preventing muscular contraction.Nerve terminal intoxication is reversible and its duration varies fordifferent BoNT serotypes.

Natural BoNTs are produced by Clostridium botulinum, and otherClostridial species such as C. butyricum, C. baratii and C. argentinenseas part of multi-protein complexes that protect the neurotoxin fromproteolytic degradation. By “botulinum neurotoxin in complex form” ismeant a botulinum neurotoxin and one or more of the proteins which arepart in nature of such multi-protein complexes (neurotoxin-associatedproteins or “NAPs”). NAPs include non-toxic non-hemagglutinin (NTNH)protein and hemagglutinin proteins (HA-17, HA-33, and HA-70). By “highpurity botulinum neurotoxin” is meant a botulinum neurotoxin essentiallyfree of NAPs.

According to an embodiment of the invention, the botulinum neurotoxin isa botulinum neurotoxin in complex form. According to another embodiment,the botulinum neurotoxin is a high purity botulinum neurotoxin.

Method for producing BoNTs through culture of natural clostridialstrains and purifying them either in complex form or high purity formare well known in the art and are described for example in Pickett,Andy. “Botulinum toxin as a clinical product: manufacture andpharmacology.” Clinical Applications of Botulinum Neurotoxin. SpringerNew York, 2014. 7-49.

High purity or essentially pure botulinum neurotoxin can be obtainedfrom a protein complex comprising botulinum toxin for example accordingto the method described in Current topics in Microbiology and Immunology(1995), 195, p. 151-154.

Alternatively, high purity botulinum neurotoxin can be produced byrecombinant expression of a BoNT gene in a heterologous host such as E.coli and purified therefrom.

Preferably, the proteinaceous neurotoxin is a botulinum neurotoxin.According to an embodiment of the invention, the botulinum neurotoxin isa botulinum neurotoxin in complex form. According to another embodiment,the botulinum neurotoxin is a high purity botulinum neurotoxin.

According to an embodiment of the invention, the botulinum neurotoxin isa botulinum neurotoxin purified from its natural clostridial strain.According to another embodiment, botulinum neurotoxin is a botulinumneurotoxin produced recombinantly in a heterologous host such as E.coli.

According to the present invention, the Botulinum neurotoxin can be aBoNT of serotype A, B, C, D, E, F or G.

According to the present invention, a botulinum neurotoxin can be amodified botulinum neurotoxin. According to the present invention, a“modified BoNT” is a BoNT which has an amino acid sequence which has atleast 50% sequence identity with SEQ ID NO 1, 2, 3, 4, 5, 6 or 7.Preferably, a modified BoNT has an amino acid sequence which has atleast 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity with SEQ ID NO 1, 2, 3, 4, 5, 6 or 7. Preferably, a modifiedBoNT is a BoNT whose amino acid sequence differs from SEQ ID NO 1, 2, 3,4, 5, 6 or 7 by less than 600, 400, 200, 150, 100, 50 or 20 amino acidsubstitutions, deletions or additions, for example by 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 amino acid substitutions, deletions or additions.

According to the present invention, a recombinant botulinum neurotoxincan be a chimeric botulinum neurotoxin. According to the presentinvention, a “chimeric BoNT” is constituted by an L, H_(N), H_(CN), andH_(CC) domain which do not all belong to the same serotype. For examplea chimeric BoNT can consist of an L chain from one serotype and a full Hchain (H_(N), H_(CN), and H_(CC) domains) from a different serotype. Achimeric BoNT can also consist of an L chain and an H_(N) domain(“LH_(N)”) from one serotype and an H_(C) domain (H_(CN) and H_(CC))from a different serotype. A chimeric BoNT can also consist of an Lchain and H_(N) and H_(CN) domains (“extended LH_(N)”) from one serotypeand an H_(CC) domain from a different serotype.

According to the invention a Light chain domain (L) can have an aminoacid sequence which has at least 50%, preferably at least 60%, 70%, 80%,90% or 95% sequence identity to one of the following amino acidsequences and which retains the ability to cleave one of the SNAREproteins involved in vesicle mediated neurotransmitter release:

-   -   Amino acid 1-448 of SEQ ID NO:1    -   Amino acid 1-440 of SEQ ID NO:2    -   Amino acid 1-441 of SEQ ID NO:3    -   Amino acid 1-445 of SEQ ID NO:4    -   Amino acid 1-422 of SEQ ID NO:5    -   Amino acid 1-439 of SEQ ID NO:6    -   Amino acid 1-441 of SEQ ID NO:7

According to the invention an H_(N) domain can have an amino acidsequence which has at least 50%, preferably at least 60%, 70%, 80%, 90%or 95% sequence identity to one of the following amino acid sequencesand which retains a translocation ability:

-   -   Amino acid 449-871 of SEQ ID NO:1    -   Amino acid 441-858 of SEQ ID NO:2    -   Amino acid 442-866 of SEQ ID NO:3    -   Amino acid 446-862 of SEQ ID NO:4    -   Amino acid 423-845 of SEQ ID NO:5    -   Amino acid 440-864 of SEQ ID NO:6    -   Amino acid 442-863 of SEQ ID NO:7

According to the invention an H_(C) domain can have an amino acidsequence which has at least 50%, preferably at least 60%, 70%, 80%, 90%or 95% sequence identity to one of the following amino acid sequencesand which retains the ability to bind to a neuromuscular cell:

-   -   Amino acid 872-1296 of SEQ ID NO:1    -   Amino acid 859-1291 of SEQ ID NO:2    -   Amino acid 867-1291 of SEQ ID NO:3    -   Amino acid 863-1276 of SEQ ID NO:4    -   Amino acid 846-1252 of SEQ ID NO:5    -   Amino acid 865-1274 of SEQ ID NO:6    -   Amino acid 864-1297 of SEQ ID NO:7

According to the invention an H_(CC) domain can have an amino acidsequence which has at least 50%, preferably at least 60%, 70%, 80%, 90%or 95% sequence identity to one of the following amino acid sequencesand which retains the ability to bind to a neuromuscular cell:

-   -   Amino acid 1111-1296 of SEQ ID NO:1    -   Amino acid 1098-1291 of SEQ ID NO:2    -   Amino acid 1112-1291 of SEQ ID NO:3    -   Amino acid 1099-1276 of SEQ ID NO:4    -   Amino acid 1086-1252 of SEQ ID NO:5    -   Amino acid 1106-1274 of SEQ ID NO:6    -   Amino acid 1106-1297 of SEQ ID NO:7

The above-identified reference sequences should be considered as aguide, as slight variations may occur according to sub-serotypes.

The “percent sequence identity” between two or more nucleic acid oramino acid sequences is a function of the number of identicalnucleotides/amino acids at identical positions shared by the alignedsequences. Thus, % identity may be calculated as the number of identicalnucleotides/amino acids at each position in an alignment divided by thetotal number of nucleotides/amino acids in the aligned sequence,multiplied by 100. Calculations of % sequence identity may also takeinto account the number of gaps, and the length of each gap that needsto be introduced to optimize alignment of two or more sequences.Sequence comparisons and the determination of percent identity betweentwo or more sequences can be carried out using specific mathematicalalgorithms, such as BLAST, which will be familiar to a skilled person.

Surfactants (or surface active agents) are compounds that are able tolower the surface tension between a liquid and a solid or between twoliquids. Surfactants can be non-ionic, anionic, cationic or amphoteric.In the compositions according to the invention, the surfactant ispreferably a non-ionic surfactant. Non-ionic surfactants includePolyoxyethylene glycol alkyl ethers, such as Octaethylene glycolmonododecyl ether or Pentaethylene glycol monododecyl ether;Polyoxypropylene glycol alkyl ethers; Glucoside alkyl ethers, such asDecyl glucoside, Lauryl glucoside or Octyl glucoside; Polyoxyethyleneglycol octylphenol ethers, such as Triton X-100; Polyoxyethylene glycolalkylphenol ethers, such as Nonoxynol-9; Glycerol alkyl esters, such asGlyceryl laurate; Polyoxyethylene glycol sorbitan alkyl esters, such asPolysorbates; Sorbitan alkyl esters, such as Spans; Cocamide MEA,cocamide DEA; Dodecyldimethylamine oxide; Block copolymers ofpolyethylene glycol and polypropylene glycol, such as Poloxamers;Polyethoxylated tallow amine (POEA).

According to a preferred embodiment, the liquid composition according tothe invention comprises a non-ionic surfactant which is a polysorbate,preferably polysorbate 20 (PS20), polysorbate 60 (PS60) or polysorbate80 (PS80). Most preferably, the non-ionic surfactant is PS80. When thesurfactant is a polysorbate, its concentration is preferably from 0.001%to 15% v/v, more preferably from 0.005 to 2% v/v, more preferably stillfrom 0.01 to 1% for example 0.01, 0.05, 0.1, 0.2, 0.5 or 1% v/v.According to one embodiment, the surfactant is PS80 at a concentrationfrom 0.05 to 0.2% v/v, for example about 0.05, 0.06, 0.07, 0.08, 0.09,0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20% v/v.

PS20 has a density of approximately 1.1 g/mL. PS60 has a density ofapproximately 1.044 g/mL. PS80 has a density of approximately 1.06 to1.09 g/mL.

Polysorbates are believed to form micelles and prevent adsorption ofproteins to surfaces and protein aggregation. Without wishing to bebound by theory, it is believed that upon degradation/oxidation,polysorbates may form peroxides and acids that may have an effect onprotein stability. Therefore, it is considered preferable that theconcentration of polysorbate be as low as possible in the formulation ofthe product. It is therefore considered preferable that theconcentration of polysorbate should not exceed 200 times its criticalmicellar concentration (CMC), more preferably it should not exceed 100,50, 20, 10 or 5 times its CMC.

For PS20 (Mw 1227.5 g/mol), the CMC is approximately 8×10⁻⁵ M at 21° C.,i.e. approximately 0.01% w/v.

For PS60 (Mw 1309 g/mol), the CMC is approximately 21×10⁻⁶ M at 21° C.,i.e. approximately 0.003% w/v.

For PS80 (Mw 1310 g/mol), the CMC is approximately 12×10⁻⁶ M at 21° C.,i.e. approximately 0.002% w/v.

According to a preferred embodiment, the polysorbate concentration isbetween 1 and 200 times its CMC at a given temperature, for exampleabout 21° C., preferably between 2 and 100 times its CMC, for exampleabout 20 or 50 times its CMC.

The liquid composition according to the invention comprises an aminoacid which is tryptophan or tyrosine. Without willing to be bound bytheory, it is hypothesized that tryptophan or tyrosine can preventoxidation of the active protein which would render it non-functional.Indeed, it is thought that the amino acid added in molar excess over theneurotoxin will be oxidized in the first place, saving the neurotoxin.It is also hypothesized that tryptophan or tyrosine can neutralizereactive degradation products of surfactants such as polysorbates.

Preferably the amino acid is tryptophan. More preferably, the amino acidis L-tryptophan.

The amino acid concentration is preferably from about 0.1 to 5 mg/mL,more preferably between 0.1 and 5 mg/mL, from 0.25 and 3 mg/mL forexample about 0.25, 0.5, 1, 1.5, 2 or 3 mg/m L.

The composition according to the invention comprises a buffer whichcomprises sodium, chloride and phosphate ions. The inventors indeedsurprisingly found that buffers without sodium, chloride and phosphateions lowered the stability of the toxin. Preferably the buffer alsocomprises potassium ions.

The buffer can for example be obtained by combining sodium chloride,potassium chloride and sodium phosphate salts. The sodium chlorideconcentration is preferably from 10 to 500 mM, preferably from about 25to 300 mM, for example about 25, 50, 75, 100, 140, 150, 200, 250 or 300mM.

The sodium phosphate concentration is preferably from 1 to 100 mM,preferably from 2 to 50 mM, for example about 2, 5, 10, 20, 30, 40 or 50mM.

The potassium chloride concentration is preferably from 1 to 50 mM,preferably from 1 to 10 mM for example about 1, 2, 3, 4, 5 or 10 mM.

The composition according to the invention has a pH between 5.5 and 8.According to a preferred embodiment, the pH is between 6.0 and 7.5, forexample about 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75,6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4,7.45 or 7.5. Preferably the pH is within one unit from physiological pH(which is around 7.4).

The composition according to the invention is liquid. The compositionpreferably comprises an aqueous diluent, more preferably water, forexample sterile water, water for injection, purified water, sterilewater for injection.

Preferably the formulation is isotonic and is suitable for injection toa patient, in particular a human patient.

The quantity of botulinum neurotoxin is commonly expressed in mouse LD50(lethal dose 50) units, defined as the median lethal intraperitonealdose in mice.

The mouse LD50 (MLD50) unit for botulinum toxins is not a standardisedunit. Indeed, assays used by different manufacturers of marketed toxinsdiffer in particular in the choice of dilution buffer. For example thetest used for Dysport® uses gelatine phosphate buffer, whereas the assayused for BOTOX® uses saline as a diluent. It is believed that gelatinebuffers protect the toxin at the high dilutions used in LD50 assays. Incontrast the use of saline as a diluent is thought to lead to some lossof potency. This could explain why when tested with the Dysport® assay,one BOTOX® unit is equivalent to approximately three units of Dysport(Straughan, D. W., 2006, ATLA 34(3), 305-313; Hambleton and Pickett,Hambleton, P., and A. M. Pickett., 1994, Journal of the Royal Society ofMedicine 87.11: 719).

Preferably, the dilution buffer used to determine the mouse LD50 is agelatine phosphate buffer. For example, the mouse LD50 can be determinedas described in Hambleton, P. et al. Production, purification andtoxoiding of Clostridium botulinum type A toxin. Eds. G. E. Jr Lewis,and P. S. Angel. Academic Press, Inc., New York, USA, 1981, p. 248.Briefly, botulinum toxin samples are serially diluted in 0.2% (w/v)gelatine 0.07M Na2HPO4 buffer at pH 6.5. Groups of mice (eg 4 to 8 miceper group) weighing about 20 g are injected intraperitoneally with asample of diluted toxin (for example 0.5 ml per animal). Dilutiongroups, for example 5 dilution groups, are selected to span the 50%lethality dose. The mice are observed for up to 96 hours and the mouselethal dose 50 (MLD50) is estimated.

The composition according to the invention preferably comprises from 4to 10000 LD50 units of botulinum neurotoxin per mL, more preferably from10 to 2000 LD50 units of botulinum neurotoxin per mL, for example 20,30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or1500 LD50 units of botulinum neurotoxin per mL.

The quantity of botulinum neurotoxin can also be expressed in ng. Thecomposition according to the invention preferably comprises from about0.01 to 75 ng of botulinum neurotoxin per mL, more preferably from about0.03 to 20 ng botulinum neurotoxin per mL, more preferably still fromabout 0.1 to 15 ng botulinum neurotoxin per mL, for example about 0.15,0.3, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ng botulinum neurotoxinper mL.

The formulation according to the invention is animal protein free. Inparticular, the compositions according to the invention comprise noalbumin, and in particular no human serum albumin. Preferably, thecomposition according to the invention is animal product free, meaningthat they comprise no constituent of animal (including human) origin.Preferably, the composition according to the invention comprises noprotein other than the proteinaceous neurotoxin. According to anotherembodiment, the composition according to the invention comprises noprotein other than the proteinaceous neurotoxin and one or more NAPs(neurotoxin-associated proteins). For the sake of doubt, it is notedamino acids are not proteins.

According to an embodiment of the invention, the composition comprisesno saccharides, including no monosaccharides, no disaccharides and nopolysaccharides.

The liquid composition according to the invention is stable over time.For example, it is stable for 2 months at 2 to 8° C. According to oneembodiment, it is stable for 3 months at 2 to 8° C., for example at 5°C. According to a preferred embodiment, it is stable for 6 months at 2to 8° C., for example at 5° C. According to one embodiment, it is stablefor 12 months at 2 to 8° C., for example at 5° C. According to oneembodiment, it is stable for 18 months at 2 to 8° C., for example at 5°C. According to one embodiment, it is stable for 24 months at 2 to 8°C., for example at 5° C. According to one embodiment, it is stable for36 months at 2 to 8° C., for example at 5° C. According to oneembodiment, it is stable for 3 months at room temperature, for exampleat 25° C. According to one embodiment, it is stable for 6 months at roomtemperature, for example at 25° C. According to one embodiment, it isstable for 2 months at 37° C.

The liquid composition according to the invention is preferably storedat a temperature between 0° and 30° C. In a preferred embodiment it isstored at 2-8° C., for example at 5° C. In another embodiment, it isstored at room temperature. Preferably it is not frozen.

Stability can be assessed through comparison of the activity of thebotulinum neurotoxin over time. Activity of the botulinum neurotoxin mayrefer to the ability of the activity of the botulinum neurotoxin to bindto its target receptor on a cell, to translocate the light chain into acell, and/or to cleave its target SNARE protein.

Methods for measuring Botulinum neurotoxin activity are well known inthe art. Botulinum neurotoxin activity can be assessed for example byusing a mouse lethality assay (LD50) as described above, a muscle tissuebased assay such as the mouse phrenic nerve hemidiaphragm assay (forexample as described in Bigalke, H. and Rummel A., Toxins 7.12(2015):4895-4905), a cell based assay (for example as described inWO201349508 or in WO2012166943) or an extracellular proteolytic activityassay such as BoTest® (Botulinum Neurotoxin Detection Kit available fromBioSentinel Inc.).

Preferably, a composition according to the invention is consideredstable if there is no more than a given percentage of loss of activityover a given period of time and at a given temperature.

According to one embodiment, a composition according to the invention isconsidered stable if there is no more than 30% loss in extracellularproteolytic activity over 3, 6, 12, 18, 24 or 36 months at 2 to 8° C.,for example no more than 30% loss in extracellular proteolytic activityover 6 months at 5° C. Preferably, a composition according to theinvention is considered stable if there is no more than 20% loss inextracellular proteolytic activity over 3 months at 5° C., morepreferably over 6, 12, 18, 24 or 36 months at 5° C. According to anotherembodiment, a composition according to the invention is consideredstable if there is no more than 40% loss in extracellular proteolyticactivity over 3 months at room temperature, for example at 25° C.Preferably, a composition according to the invention is consideredstable if there is no more than 30% loss in extracellular proteolyticactivity over 3 months at 25° C., more preferably over 6 months at 25°C. According to another embodiment, a composition according to theinvention is considered stable if there is no more than 50% loss inextracellular proteolytic activity over 2 months at 37° C. Extracellularproteolytic activity can be measured with the BoTest® assay.

According to one embodiment, a composition according to the invention isconsidered stable if there is no more than 30% loss in MLD50 units over2, 3, 6, 12, 18, 24 or 36 months at 2 to 8° C., for example no more than30% loss in MLD50 units over 6 months at 5° C. Preferably, a compositionaccording to the invention is considered stable if there is no more than20% loss in MLD50 units over 2 months at 5° C., more preferably over 3,6, 12, 18, 24 or 36 months at 5° C. According to another embodiment, acomposition according to the invention is considered stable if there isno more than 40% loss in MLD50 units over 2 or 3 months at roomtemperature, for example at 25° C. Preferably, a composition accordingto the invention is considered stable if there is no more than 30% lossin MLD50 units over 3 months at 25° C., more preferably over 6 months at25° C. According to another embodiment, a composition according to theinvention is considered stable if there is no more than 50% loss inMLD50 units over 2 months at 37° C. MLD50 units can be measured asindicated above.

The liquid compositions according to the invention can be stored insealed vials or syringes, for example glass vials or syringes,preferably type 1 (or “body neutral”) glass vials or syringes.Preferably there is no or very little oxygen in the vial or syringe. Thevials or syringes can for example be filled in an atmosphere with anoxygen below 100 ppm, preferably below 50 ppm, and nitrogen gas can beused as a protective atmosphere in the vials. When glass vials are used,they can for example be capped with chlorobutyl or bromobutyl rubberstoppers, which can be FluroTec® coated stoppers. Preferably, the liquidcompositions according to the invention are stored in glass vials cappedwith FluroTec® coated stoppers.

According to one embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   4 to 10000 LD50 units of botulinum neurotoxin per mL,    -   0.001 to 15% v/v polysorbate,    -   0.1 to 5 mg/mL tryptophan,    -   10 to 500 mM NaCl,    -   1 to 50 mM KCl,    -   1 to 100 mM Sodium phosphate,    -   has a pH between 5.5 and 8, and is stable for 6 months at 5° C.

According to one embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   10 to 2000 LD50 units of botulinum neurotoxin per mL,    -   0.005 to 2% v/v polysorbate,    -   0.1 to 5 mg/mL tryptophan,    -   25 to 300 mM NaCl,    -   1 to 10 mM KCl,    -   2 to 50 mM Sodium phosphate,    -   has a pH between 6.0 and 7.5, and is stable for 12 months at 5°        C.

According to one embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   10 to 2000 LD50 units of botulinum neurotoxin per mL,    -   0.05 to 0.2% v/v polysorbate 80,    -   0.1 to 5 mg/mL tryptophan,    -   25 to 300 mM NaCl,    -   1 to 10 mM KCl,    -   2 to 50 mM Sodium phosphate,    -   has a pH between 6.0 and 7.5, and is stable for 12 months at 5°        C.

According to one embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.2% v/v polysorbate 80,    -   1 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.6.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.04% v/v polysorbate 80,    -   1 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.9.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin B,    -   0.25% v/v polysorbate 20,    -   4 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 7.4.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.01% v/v polysorbate 80,    -   0.25 mg/mL tryptophan    -   255 mM NaCl,    -   2 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 7.2.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.01% v/v polysorbate 80,    -   0.25 mg/mL tryptophan    -   255 mM NaCl,    -   10 mM KCl,    -   50 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.3.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   1% v/v polysorbate 80,    -   0.25 mg/mL tryptophan    -   255 mM NaCl,    -   50 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.3.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   1% v/v polysorbate 80,    -   3 mg/mL tryptophan    -   255 mM NaCl,    -   10 mM KCl,    -   50 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 7.2.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.1% v/v polysorbate 80,    -   1.625 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.75.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.01% v/v polysorbate 80,    -   1 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.75.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   0.1% v/v polysorbate 80,    -   1 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.75.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin A,    -   1% v/v polysorbate 80,    -   1 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 6.75.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin B,    -   15% v/v polysorbate 20,    -   1 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 7.4.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin B,    -   15% v/v polysorbate 20,    -   4 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 7.4.

According to another embodiment, a liquid composition according to theinvention comprises or consists essentially of:

-   -   Botulinum neurotoxin B,    -   0.25% v/v polysorbate 20,    -   4 mg/mL tryptophan    -   140 mM NaCl,    -   3 mM KCl,    -   10 mM Sodium phosphate,    -   wherein the pH of said composition is approximately 7.4.

Another aspect is the use of the liquid compositions according to theinvention in therapy.

The liquid compositions according to the invention can be used intherapy to treat or prevent muscular disorders, neuromuscular disorders,neurological disorders, ophtalmological disorders, pain disorders,psychological disorders, articular disorders, inflammatory disorders,endocrine disorders or urological disorders.

For example, the liquid compositions according to the invention can beused for treating or preventing a disease, condition or syndromeselected from the following:

-   -   ophtalmological disorders selected from the group consisting of        blepharospasm, strabismus (including restrictive or myogenic        strabismus), amblyopia, oscillopsia, protective ptosis,        therapeutic ptosis for corneal protection, nystagmus, estropia,        diplopia, entropion, eyelid retraction, orbital myopathy,        heterophoria, concomitant misalignment, nonconcomitant        misalignment, primary or secondary esotropia or exotropia,        internuclear ophthalmoplegia, skew deviation, Duane's syndrome        and upper eyelid retraction;    -   movement disorders including hemifacial spasm, torticollis,        spasticity of the child or of the adult (e.g. in cerebral palsy,        post-stroke, multiple sclerosis, traumatic brain injury or        spinal cord injury patients), idiopathic focal dystonias, muscle        stiffness, Writer's cramp, hand dystonia, VI nerve palsy,        oromandibular dystonia, head tremor, tardive dyskinesia, tardive        dystonia, occupational cramps (including musicians' cramp),        facial nerve palsy, jaw closing spasm, facial spasm, synkinesia,        tremor, primary writing tremor, myoclonus,        stiff-person-syndrome, foot dystonia, facial paralysis,        painful-arm-and-moving-fingers-syndrome, tic disorders, dystonic        tics, Tourette's syndrome, neuromyotonia, trembling chin,        lateral rectus palsy, dystonic foot inversion, jaw dystonia,        Rabbit syndrome, cerebellar tremor, III nerve palsy, palatal        myoclonus, akasthesia, muscle cramps, IV nerve palsy,        freezing-of-gait, extensor truncal dystonia, post-facial nerve        palsy synkinesis, secondary dystonia, Parkinson's disease,        Huntington's chorea, epilepsy, off period dystonia, cephalic        tetanus, myokymia and benign cramp-fasciculation syndrome;    -   otorhinolaryngological disorders including spasmodic dysphonia,        otic disorders, hearing impairment, ear click, tinnitus,        vertigo, Meniere's disease, cochlear nerve dysfunction,        stuttering, cricopharyngeal dysphagia, bruxism, closure of        larynx in chronic aspiration, vocal fold granuloma, ventricular        dystonia, ventricular dysphonia, mutational dysphonia, trismus,        snoring, voice tremor, aspiration, tongue protrusion dystonia,        palatal tremor, deep bite of lip and laryngeal dystonia; First        Bite Syndrome;    -   gastrointestinal disorders including achalasia, anal fissure,        constipation, temperomandibular joint dysfunction, sphincter of        Oddi dysfunction, sustained sphincter of Oddi hypertension,        intestinal muscle disorders, puborectalis syndrome, anismus,        pyloric spasm, gall bladder dysfunction, gastrointestinal or        oesophageal motility dysfunction, diffuse oesophageal spasm and        gastroparesis;    -   urogenital disorders including detrusor sphincter dyssynergia,        detrusor hyperreflexia, neurogenic bladder dysfunction (e.g. in        Parkinson's disease, spinal cord injury, stroke or multiple        sclerosis patients), overactive bladder, neurogenic detrusor        overactivity, bladder spasms, urinary incontinence, urinary        retention, hypertrophied bladder neck, voiding dysfunction,        interstitial cystitis, vaginismus, endometriosis, pelvic pain,        prostate gland enlargement (Benign Prostatic Hyperplasia),        prostatodynia, prostate cancer and priapism;    -   dermatological disorders including cutaneous cell proliferative        disorders, skin wounds, psoriasis, rosacea, acne; rare        hereditary skin disorders such as Fox-Fordyce syndrome or        Hailey-Hailey disease; keloid and hypertrophic scar reduction;        pore size reduction; inflammatory conditions of the skin;        painful inflammatory conditions of the skin;    -   pain disorders including back pain (upper back pain, lower back        pain), myofascial pain, tension headache, fibromyalgia, painful        syndromes, myalgia, migraine, whiplash, joint pain,        post-operative pain, pain not associated with a muscle spasm and        pain associated with smooth muscle disorders;    -   inflammatory disorders including pancreatitis, neurogenic        inflammatory disorders (including gout, tendonitis, bursitis,        dermatomyositis and ankylosing spondylitis);    -   secretory disorders such as excessive gland secretions,        hyperhidrosis (including axillary hyperhidrosis, palmar        hyperhidrosis and Frey's syndrome), hypersalivation,        sialorrhoea, bromhidrosis, mucus hypersecretion,        hyperlacrimation, holocrine gland dysfunction; excess sebum        secretion;    -   respiratory disorders including rhinitis (including allergic        rhinitis), COPD, asthma and tuberculosis;    -   hypertrophic disorders including muscle enlargement, masseteric        hypertrophy, acromegaly and neurogenic tibialis anterior        hypertrophy with myalgia;    -   articular disorders including tennis elbow (or epicondilytis of        the elbow), inflammation of joints, coxarthrosis,        osteoarthritis, rotator muscle cap pathology of the shoulder,        rheumatoid arthritis and carpal tunnel syndrome;    -   endocrine disorders like type 2 diabetes, hyperglucagonism,        hyperinsulinism, hypoinsulinism, hypercalcemia, hypocalcemia,        thyroid disorders (including Grave's disease, thyroiditis,        Hashimoto's thyroiditis, hyperthyroidism and hypothyroidism),        parathyroid disorders (including hyperparathyroidism and        hypoparathyroidism), Gushing's syndrome and obesity;    -   autoimmune diseases like systemic lupus erythemotosus;    -   proliferative diseases including paraganglioma tumors, prostate        cancer and bone tumors;    -   traumatic injuries including sports injuries, muscle injuries,        tendon wounds and bone fractures; and    -   veterinary uses (e.g. immobilisation of mammals, equine colic,        animal achalasia or animal muscle spasms).

The liquid compositions according to the invention can also be used inaesthetic medicine (that is for improving cosmetic appearance), inparticular for treating or preventing skin wrinkles, in particularfacial wrinkles such as facial frown lines, wrinkles of the contour ofthe eye, glabellar frown lines, downturned mouth, wrinkles of the neck(platysmal bands), wrinkles of the chin (mentalis, peau d'orange,dimpled chin), forehead lines, “scratched skin” wrinkles, nasal lifttreatment or sleep lines. According to this aspect of the invention, thesubject to be treated or prevented for improving cosmetic appearance ispreferably not suffering from any of the disorders, conditions orsyndromes that are described above. More preferably, said subject is ahealthy subject (i.e. not suffering from any disease, condition orsyndrome).

The liquid compositions according to the invention can be used incombination with another therapeutic compound. In one embodiment theliquid compositions according to the invention is administered incombination with an analgesic compound for treating pain, in particularin combination with an opioid derivative such as morphine as describedin WO 2007/144493 the content of which is herein incorporated byreference. In another embodiment, the liquid compositions according tothe invention is administered in combination with hyaluronic acid, forexample for treating prostate cancer as described in WO 2015/0444416 thecontent of which is herein incorporated by reference.

A further aspect of the present invention is the use of an amino acidselected from tryptophan and tyrosine to protect a proteinaceousneurotoxin from degradation in a liquid composition which is free ofanimal derived proteins.

According to a preferred embodiment, the amino acid is tryptophan, morepreferably L-tryptophan.

Preferably, the proteinaceous neurotoxin is a botulinum neurotoxin.According to an embodiment of the invention, the botulinum neurotoxin isa botulinum neurotoxin in complex form. According to another embodiment,the botulinum neurotoxin is a high purity botulinum neurotoxin.According to an embodiment of the invention, the botulinum neurotoxin isa botulinum neurotoxin purified from its natural clostridial strain.According to another embodiment, botulinum neurotoxin is a botulinumneurotoxin produced recombinantly in a heterologous host such as E.coli. According to the present invention, the Botulinum neurotoxin canbe a BoNT of serotype A, B, C, D, E, F or G. According to the presentinvention, a botulinum neurotoxin can be a modified botulinum neurotoxinas described above. According to the present invention, a recombinantbotulinum neurotoxin can be a chimeric botulinum neurotoxin as describedabove.

According to a preferred embodiment, the amino acid is used incombination with a surfactant and a buffer comprising sodium, chlorideand phosphate ions, and the liquid composition has a pH between 5.5 and8. Preferably, the surfactant is a non-ionic surfactant, more preferablya polysorbate, for example PS20, PS60 or PS80. Most preferably, thenon-ionic surfactant is PS80. Preferably, the buffer also comprisespotassium ions. The buffer can for example be obtained by combiningsodium chloride, potassium chloride and sodium phosphate salts.According to a preferred embodiment, the pH is between 6.0 and 7.5, forexample 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8,6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45or 7.5. Preferably the pH is within one unit from physiological pH(which is around 7.4).

According to a preferred embodiment of the use according to theinvention, the liquid composition is stable for 2 months. For example,it is stable for 2 months at 2 to 8° C. According to one embodiment, itis stable for 3 months at 2 to 8° C., for example at 5° C. According toa preferred embodiment, it is stable for 6 months at 2 to 8° C., forexample at 5° C. According to one embodiment, it is stable for 12 monthsat 2 to 8° C., for example at 5° C. According to one embodiment, it isstable for 18 months at 2 to 8° C., for example at 5° C. According toone embodiment, it is stable for 24 months at 2 to 8° C., for example at5° C. According to one embodiment, it is stable for 36 months at 2 to 8°C., for example at 5° C. According to one embodiment, it is stable for 3months at room temperature, for example at 25° C. According to oneembodiment, it is stable for 6 months at room temperature, for exampleat 25° C.

EXAMPLES

1. Preparation of Stable Liquid Botulinum Toxin A Formulations

Liquid botulinum toxin preparations containing 15 ng/mL of highlypurified BoNT/A, 15% v/v polysorbate 20, an amino acid selected fromtyrosine (Tyr), tryptophan (Trp) and cysteine (Cys) or a mixture ofmethionine (Met), tyrosine (Tyr), tryptophan (Trp) and cysteine (Cys)(Sigma Aldrich), and Phosphate Buffer Saline (PBS from Calbiochem) (140mM NaCl, 10 mM sodium phosphate and 3 mM KCl at pH 7.4 at 25° C.) wereprepared, filtered using 0.22 μm PVDF (polyvinylidenflourid) filters andstored in siliconized 2 mL glass syringes for 6 days at 40° C., afterwhich a potency test was performed for each preparation.

For the potency test, the syringes containing the preparations wereemptied in 2 mL glass vials (Chromacol, Gold) with lids containing PTFEtreated rubber septa (Chromacol) or in 1.7 mL plastic micro centrifugetubes (Axygen, Maximum Recovery) which both have low protein adsorptionproperties. The preparations were subsequently diluted using 0.9% NaClsolution with 3% human serum albumin (HSA). For each preparation, 50 μLof sample was injected into the Gastrocnemius muscle of mice on the sameday as the dilution was performed. The mice were monitored for 3 daysand the degree of paralysis was recorded.

The results are shown in table 1.

TABLE 1 an accelerated storage test (6 days at 40° C.) of amino acidadditions on BoNT/A stability. Potency test Formulation Dilution in 0.9Inj. BoNT/A NaCl with 3% Dose Potency rating Buffer conc. Amino acid HSA(times) (ng) Day 1 Day 2 Day 3 PBS pH 7.4 15 ng/mL Trp 0.25 mg/mL +  2 x0.25 — — WN 15% Cys 0.25 mg/mL + 20 x 0.025 — — WN polysorbate Met 0.25mg/mL + 20 Tyr 0.25 mg/mL Cys 1 mg/mL  2 x 0.25 — — WN 20 x 0.025 — — WNTyr 0.74 mg/mL  2 x 0.25 — — Sharp PA whole abdomen 20 x 0.025 — — WNTrp 1 mg/mL  2 x 0.25 — † 20 x 0.025 — — WN —  2 x 0.25 — — WN 20 x0.025 — — WN Paralysis results in mice: — = not analysed, WN = withoutnote, PA = paralysis and † = death.

Tyrosine and tryptophan were found to have a protective effect againstBoNT/A degradation. Tryptophan was found to have the strongestprotective effect. Cysteine, as well as the mixture containing all 4amino acids did not have a protective effect.

2. Preparation of a Stable Liquid Botulinum Toxin B Formulation

Liquid botulinum toxin preparations containing 350 ng/mL of highlypurified BoNT/B, 15% v/v polysorbate 20, an amino acid selected fromtyrosine (Tyr), tryptophan (Trp) and cysteine (Cys) or a mixture ofmethionine (Met), tyrosine (Tyr), tryptophan (Trp) and cysteine (Cys),and Phosphate Buffer Saline (PBS) at pH 7.4 were prepared, filteredusing 0.22 μm filters and stored in siliconized 2 mL glass syringes fortwo weeks at 40° C., after which a potency test was performed for eachpreparation as described above.

The results are shown in table 2.

TABLE 2 an accelerated storage test (two weeks at 40° C.) of amino acidadditions on BoNT/B stability. Potency test Formulation Dilution in 0.9Inj. BoNT/B NaCl with 3% Dose Potency rating Buffer conc. Amino acid HSA(times) (ng) Day 1 Day 2 Day 3 PBS pH 7.4 350 ng/mL Trp 0.25 mg/mL + 10x 1.75 — — PA 15% Cys 0.25 mg/mL + polysorbate Met 0.25 mg/mL + 20 Tyr0.25 mg/mL Cys 1 mg/mL 10 x 1.75 — — PA Tyr 0.575 mg/mL 10 x 1.75 — †Trp 1 mg/mL 10 x 1.75 — † 35 x 0.5 — — PA Paralysis results in mice: — =not analysed, WN = without note, PA = paralysis and † = death.

Tyrosine and tryptophan were found to have a protective effect againstBoNT/B degradation. Cysteine, as well as the mixture containing all 4amino acids also had a protective effect but to a weaker extent.

3. Evaluation of Different Concentrations of Tryptophan and Polysorbate20

Liquid botulinum toxin preparations containing highly purified BoNT/A orBoNT/B and various concentrations of polysorbate 20 (PS 20) andtryptophan and Phosphate Buffer Saline (PBS) at pH 7.4 were prepared,filtered using 0.22 μm filters and stored in siliconized 2 mL glasssyringes. Hind limb paralysis potency tests were performed for eachpreparation as described above.

The results are shown in table 3.

TABLE 3 evaluation of Trp and polysorbate 20 (PS20) concentrations onBoNT/A or BoNT/B stability. POTENCY TESTING Potency rating (1-3 d) forsamples stored at different Formulation temperatures and lengths BoNTDilution in 0.9 Inj. 5° C. 25° C. conc. Trp PS20 NaCl with 3% Dose 6months 5 weeks 4 months BoNT (ng/mL) (mg/mL) (%) HSA (times) (ng) 1 d 2d 3 d 1 d 2 d 3 d 1 d 2 d 3 d BoNT/A 15 8 15 1 x 0.75 — † — — † — — PA 5x 0.15 — — † — — † — — WN 15 8 0.25 1 x 0.75 — † — —  †* — — † 5 x 0.15— — † —  †* — —  WN² 15 1 15 1 x 0.75 — † — † † 5 x 0.15 — — † — — † — † PA³ BoNT/B 100 8 15 1 x 5 — — † — — † — — PA 10 x  0.5 — — PA¹ — — PA —— PA 100 8 0.25 1 x 5 — — † — — † † 10 x  0.5 — — PA⁴ — — WN¹ — — PA 1001 15 1 x 5 — — † — — † † 10 x  0.5 — — PA⁴ — PA — — PA Paralysis resultsin mice: — = not analysed, WN = without note, PA = paralysis and t =death. Paralysis degree (PA): ¹Toes affected; ²Slightly numb in hindleg; ³Both hind legs paralysed; ⁴Hind leg paralysed; Elution buffer frompurification (5): 50 mM sodium acetate pH 4.5 with 0.2% (v:v)polysorbate 20 and 400 mM sodium chloride. *a mix up of two BoNT/Adilutions of the 25° C. 8 mg/mL Trp 0.25% polysorbate 20 has probablyoccurred.

4. Evaluation of Different Salt Concentrations in BoNT/B Preparations

Liquid botulinum toxin preparations containing 100 ng/mL of highlypurified BoNT/B, polysorbate 20, tryptophan from various amino acidsuppliers and a buffer selected from PBS pH 7.4 (Calbiochem), 12 nMphosphate buffer pH 7 (Apoteket) and 20 mM sodium acetate (NaAc) pH 5.5(NaAc from Fluka and acetic acid from Merck) were prepared, filteredusing 0.22 μm filters and stored in siliconized 2 mL glass syringes.Hind limb paralysis potency tests were performed for each preparation asdescribed above.

The results are shown in table 4.

TABLE 4 evaluation of different salt concentrations on stability ofBoNT/B. Potency rating (1-3 d) for samples stored at Potency testingdifferent length BoNT/B Trp Dilution in 0.9 Inj. 40° C. conc.manufacturer PS20 NaCl with 3% Dose 2 weeks 5 weeks (ng/mL) Buffer andconc. (%) HSA (times) (ng) 1 d 2 d 3 d 1 d 2 d 3 d 100 PBS Ajinomoto 15 1 x 5 — † pH 7.4 4 mg/mL 10 x 0.5 — — PA 100 PBS Sigma 15  1 x 5 — † pH7.4 Aldrich 10 x 0.5 — — PA 4 mg/mL 100 PBS Sigma 15  1 x 5 — † pH 7.4Aldrich 10 x 0.5 — — PA 1 mg/mL 100 PBS Sigma 0.25  1 x 5 — † — † pH 7.4Aldrich 10 x 0.5 — — PA — — PA² 4 mg/mL 100 12 mM Sigma 15  1 x 5 — — WNPhosphate Aldrich 10 x 0.5 — —  WN¹ pH 7 4 mg/mL 100 20 mM Sigma 15  1 x5 — — PA NaAc Aldrich 10 x 0.5 — — PA pH 5.5 4 mg/mL Paralysis resultsin mice: — = not analysed, WN = without note, PA = paralysis and † =death. ¹Some loss of function; ²Weak paralysis

The results show that the preparations containing the PBS buffer(containing sodium, chloride, phosphate and potassium ions) appears toplay a role in the stability of the botulinum toxin.

5. Evaluation of Different Stabilizers

Liquid botulinum toxin preparations containing 15 ng/mL of highlypurified BoNT/A, a polysorbate 20 (PS20) or polysorbate 80 (PS80) orHSA, tryptophan and PBS were prepared, filtered using 0.22 μm filtersand stored in siliconized 2 mL glass syringes. Hind limb paralysispotency tests were performed for each preparation as described above.

The results are shown in table 5.

TABLE 5 evaluation of different surfactants on stability of BoNT/A.Potency rating (1-3 d) for samples stored at different Potency testinglength BoNT/A Dilution in 0.9 Inj. 40° C. conc. NaCl with 3% Dose 6 days4 weeks 3 months (ng/mL) Buffer Trp Stabiliser HSA (times) (ng) 1 d 2 d3 d 1 d 2 d 3 d 1 d 2 d 3 d 15 PBS 1 mg/mL PS 80 1.7 x 0.45 † † — — PA³pH 7.4 0.25%  15 x 0.05 † † — — WA 15 PBS 1 mg/mL PS 20 1.7 x 0.45 † † —— PA² pH 7.4 0.25%  15 x 0.05 † — — PA — — WN 15 PBS — HSA 1.7 x 0.45 †† — † pH 7.4 1 mg/mL  15 x 0.05 † † — — PA¹ Paralysis results in mice: —= not analysed, WN = without note, PA = paralysis and † = death. ¹Severeparalysis both hind legs; ²Angles paws; ³Severe paralysis

6. Evaluation of Different Formulations

Liquid botulinum toxin preparations containing 10 ng/mL of highlypurified BoNT/A, 0.25% PS80, 1 mg/mL tryptophan and PBS were prepared asdescribed above. The pH was adjusted to 6.6 and 7.0 by adding HCl. Eachpreparation was stored 5 weeks at 40° C.

Each preparation was then diluted 10 times and hind limb paralysispotency tests were performed as described above (0.05 ng per injection).In both cases, hind limb paralysis was observed at day 3. The paralysiswas stronger with the pH 6.6 preparation.

7. Evaluation of Different Formulations

Liquid botulinum toxin preparations containing 0.3 ng/mL of highlypurified BoNT/A, a polysorbate selected from PS20 and PS80, 1 mg/mLtryptophan and 12 mM PBS at pH 7.4 were prepared as described above. ThepH of each preparation was adjusted to pH 6.6 or 6.9 by adding 1.2 MHCl.

Polysorbate 20 was tested at one concentration, 0.2% w/v, correspondingto about 20 times its CMC (critical micellar concentration, about 0.01%w/v at 21° C.). Polysorbate 80 was tested 0.04% and 0.2% w/v,corresponding respectively to about 20 and 100 times its CMC (about0.002% w/v at 21° C.).

TABLE 6 Choice of polysorbate and pH Composition PS20 % w/v PS20 % w/vpH PS20-1 0.2 — 6.6 PS20-2 0.2 — 6.9 PS80-1 — 0.04 6.6 PS80-2 — 0.04 6.9PS80-3 — 0.2 6.6

For each preparation, a volume of 0.5 mL was filled in 1 mL long glasssyringes (BD) and sealed with a fluorocarbon coated plunger.

The potency was measured by hind limb paralysis test on mice asdescribed above.

No decrease in potency was observed in any formulation after 6 monthsstorage at 5° C. and after 25° C.

8. Evaluation of Different Formulations

Nineteen different formulations containing highly purified botulinumneurotoxin type A were prepared with varying concentrations ofpolysorbate 80, tryptophan, sodium phosphate, sodium chloride, potassiumchloride and varying pH. Each formulation had a target nominal potencyof 500 U/mL. Each formulation was degassed, filtered through 0.2 μmfilter and filled into vials. Nitrogen gas was used as a protectiveatmosphere in the vials. The filling was performed in an anaerobicchamber. Each formulation was filled in 1 mL aliquots in a nitrogenatmosphere in 2 mL glass vials capped with FluroTec® stoppers sealedwith aluminium flip off seals and stored upright.

The stability of the 19 formulations was assessed at 5° C., 25° C. and37° C. using BoTest® to measure potency.

TABLE 7 Excipient compositions Sodium Polysorbate Exp phosphateTryptophan 80 NaCl KCl Name pH (mM) (mg/mL) (v %) (mM) (mM) N1 6.3 20.25 0.01 25 0 N2 7.2 2 0.25 0.01 255 0 N3 6.3 50 0.25 0.01 255 10 N47.2 50 0.25 0.01 25 10 N5 6.3 2 3 0.01 255 10 N6 7.2 2 3 0.01 25 10 N76.3 50 3 0.01 25 0 N8 7.2 50 3 0.01 255 0 N9 6.3 2 0.25 1 25 10 N10 7.22 0.25 1 255 10 N11 6.3 50 0.25 1 255 0 N12 7.2 50 0.25 1 25 0 N13 6.3 23 1 255 0 N14 7.2 2 3 1 25 0 N15 6.3 50 3 1 25 10 N16 7.2 50 3 1 255 10N17 6.75 10 1.625 0.1 140 3

TABLE 8 Packaging components Article Article number, Supplier Clearglass vial of boro silicate Type I plus, 1097221, Schott 2 mL GreyFlurotec coated bromobutyl 1356 4023/50, West stopper Westar RS, 13 mmAluminium flip off seals, 13 mm 5920-6623, West

For all formulations the solution remained clear and for most partscolourless.

The excipients concentrations tested in this study seem not to affectthe pH of the formulations during the time interval tested. The potencyresults are presented in table 9.

TABLE 9 Botest potency results Baseline potency Remaining potencycompared to base line (BoTest) (U/mL) (Botest) 2 months 37° C. 6 months25° C. 6 months 5° C. pH 0 month U/mL % U/mL % U/mL % N1 6.3 86 0 0 0 039 45 N2 7.2 474 210 44 389 82 485 102 N3 6.3 449 288 64 406 90 512 114N4 7.2 299 62 21 368 123 406 136 N5 6.3 378 263 70 385 102 422 112 N67.2 93 0 0 0 0 52 56 N7 6.3 238 0 0 0 0 239 100 N8 7.2 375 305 81 402107 450 120 N9 6.3 196 0 0 100 51 294 150 N10 7.2 354 201 57 408 115 411116 N11 6.3 438 197 45 372 85 492 112 N12 7.2 411 96 23 295 72 417 101N13 6.3 304 185 61 403 133 416 137 N14 7.2 206 0 0 0 0 183 89 N15 6.3197 155 79 231 117 214 109 N16 7.2 476 111 48 390 82 685 144 N17 6.75402 250 62 286 71 488 121

For several compositions there was no more than 30% loss in potency over6 months at 5° C. and/or no more than about 40% loss in potency over 3months at 25° C. and/or no more than about 50% loss in potency over 2months at 37° C.

9. Evaluation of PS 60

A formulation containing highly purified botulinum neurotoxin type A wasprepared with 0.1% (v/v) PS60, 1 mg/mL L-Tryptophan, 10 mM sodiumphosphate, 140 mM sodium chloride, 3 mM potassium chloride and water forinjection. The pH was adjusted to 6.75 with HCl. The formulation had atarget nominal potency of 100 U/mL. The formulation was degassed,filtered through 0.2 μm filters and filled into 2 mL vials asepticallyin an anaerobic chamber with nitrogen atmosphere with a fill volume of 1mL. Nitrogen gas was used as protective atmosphere in the vials. Thevials were capped with FluroTec® stoppers sealed with aluminium flip offseals.

TABLE 10 Packaging components Article Article number, Supplier Clearglass vial of boro silicate 1097221, Schott Type I plus, 2 mL 13 mm Injstopper coated bromobutyl INJ13TB3WRS, Nordic Pack 4023-50 grey Bluealuminium flip off seals, 13 mm 5920-1164, Nordic Pack

The potency over time at 37° C. and 25° C. was measured by the MLD50test as described herein.

At 37° C., the remaining potency after 9 weeks was around 50-55% of theinitial potency.

At 25° C., the remaining potency after 3 months was about 80% of theinitial potency.

1. A liquid composition comprising a proteinaceous neurotoxin, asurfactant, an amino acid selected from tryptophan and tyrosine, abuffer comprising sodium, chloride and phosphate ions, wherein saidliquid composition has a pH between 5.5 and 8, wherein said compositionis free of animal derived proteins, and wherein said liquid compositionis stable over time.
 2. A liquid composition according to claim 1,wherein said surfactant is a non-ionic surfactant.
 3. A liquidcomposition according to claim 2, wherein said non-ionic surfactant is apolysorbate, preferably Polysorbate 20, Polysorbate 60 or Polysorbate80.
 4. A liquid composition according to claim 1, wherein said aminoacid is tryptophan, preferably L-tryptophan.
 5. A liquid compositionaccording to claim 1, wherein said buffer further comprises potassiumions.
 6. A liquid composition according to claim 1, wherein saidcomposition has a pH between 6.0 and 7.5.
 7. A liquid compositionaccording to claim 1, wherein no more than 30% loss in extracellularproteolytic activity occurs over 2, 3, 6, 12, 18, 24 or 36 months at 5°C.
 8. A liquid composition according to claim 1, wherein saidproteinaceous neurotoxin is a botulinum neurotoxin, selected from anatural botulinum neurotoxin in complex form, a high purity naturalbotulinum neurotoxin and a recombinant botulinum neurotoxin.
 9. A liquidcomposition according to claim 8, wherein said botulinum neurotoxin is arecombinant botulinum neurotoxin selected from a botulinum neurotoxin A,B, C, D, E, F or G, a modified botulinum neurotoxin and a chimericbotulinum neurotoxin.
 10. A liquid composition according to claim 1,wherein said liquid composition comprises: 4 to 10000 LD50 units ofbotulinum neurotoxin per mL, 0.001 to 15% v/v polysorbate, 0.1 to 5mg/mL tryptophan, 10 to 500 mM NaCl, 1 to 50 mM KCl, 1 to 100 mM Sodiumphosphate, has a pH between 5.5 and 8, and is stable for 6 months at 5°C.
 11. A liquid composition according to claim 10, wherein said liquidcomposition comprises: 10 to 2000 LD50 units of botulinum neurotoxin permL, 0.05 to 0.2% v/v polysorbate 80, 0.1 to 5 mg/mL tryptophan, 25 to300 mM NaCl, 1 to 10 mM KCl, 2 to 50 mM Sodium phosphate, has a pHbetween 6.0 and 7.5, and is stable for 12 months at 5° C.
 12. A liquidcomposition according to claim 1 for use in therapy.
 13. A liquidcomposition according to claim 12 for use in treating or preventingmuscular disorders, neuromuscular disorders, neurological disorders,ophthalmological disorders, pain disorders, psychological disorders,articular disorders, inflammatory disorders, endocrine disorders orurological disorders.
 14. Use of liquid composition according to claim 1in aesthetic medicine, for example for treating or preventing skinwrinkles, in particular facial wrinkles such as facial frown lines,wrinkles of the contour of the eye, glabellar frown lines, downturnedmouth, wrinkles of the neck (platysmal bands), wrinkles of the chin(mentalis, peau d′ orange, dimpled chin), forehead lines, “scratchedskin” wrinkles, nasal lift treatment or sleep lines.
 15. Use of an aminoacid selected from tryptophan and tyrosine to protect a proteinaceousneurotoxin from degradation in a liquid composition which is free ofanimal derived proteins.
 16. Use according to claim 15, wherein saidamino acid is tryptophan.
 17. Use according to claim 15, wherein saidproteinaceous neurotoxin is a botulinum neurotoxin.
 18. Use according toclaim 15, wherein said amino acid is used in combination with asurfactant and a buffer comprising sodium, chloride and phosphate ions,and said liquid composition has a pH between 5.5 and 8.